Acute inflammatory responses are highly beneficial for host defense to eliminate infections and to initiate wound healing. However, inappropriate, uncontrolled inflammatory responses cause the detrimental pathologies of an expanding spectrum of inflammatory diseases. A key pathway promoting inflammatory responses is the inflammasome, which is a signaling platform composed of an upstream sensor of the NLR or ALR families, the adaptor ASC and Caspase-1. Inflammasome activation promotes activation of Caspase-1 and subsequent release of interleukin (IL)-1b and IL-18 and the induction of pyroptotic cell death. Inflammasomes are activated by a 2-step mechanism involving priming and activation, but the precise regulatory mechanisms that control and maintain a balanced inflammasome response and consequently homeostasis, are still poorly understood, but are key for developing novel and improved therapies. The research outlined in this renewal application is focused on elucidating the molecular mechanism by which two by us discovered inhibitors of this response function to dampen inflammation, and how this activity is important to prevent inflammatory disease. We discovered all three members of the PYRIN domain (PYD)- only protein (POP) family of small endogenous proteins, which we demonstrated to function by inhibiting and resolving inflammatory responses. POPs very recently evolved in humans, but are lacking from mice and we generated novel transgenic mouse models to study POPs in macrophages in vivo. During the 1st funding period, we discovered the precise mechanism by which each of these POPs inhibit inflammasome activation at the level of inflammasome assembly. We also discovered that two members have a second unique inflammasome-independent role in regulating priming of macrophages, which is also the first key step in inflammasome activation. Our main goal for this renewal application is to delineate the precise mechanisms by which POP2 and POP3 regulate inflammatory priming of macrophages, employing biochemical, molecular and genetic studies, focusing on type I interferon (IFN-I) production and non-canonical NF-kB activation. Hence, POPs regulate inflammatory responses of macrophages at several levels, which ultimately prevents cytokine release. Therefore, dissecting these unique POP2 and POP3 activities will provide novel insights into how this important, but still poorly understood protein family regulates key innate immune signaling pathways, which is therefore highly significant for better understanding inflammatory disease pathologies. Collectively, our results will therefore have tremendous implications for human health.